Molecular bases of thermal denaturation and aggregation of whey protein have been extensively researched. When heated to a temperature above about 65° C., whey protein denatures and exposes hydrophobic amino acids originally imbedded at the globular state, facilitating intermolecular attraction. The possibility of intermolecular aggregation depends mostly on electrostatic interactions, hydrophobic interactions, and intra- and intermolecular disulfide bonds via sulfhydryl-disulfide interchange. The significance of these physical and chemical forces to protein aggregation is manipulated by the thermodynamic state (native, partially or completely denatured molecular structures), pH, and ionic strength and type. In addition, thermal aggregation of whey protein is influenced by cosolutes such as glycerol, sorbitol, and sucrose that may also co-exist in certain food matrices. Co-solutes may enhance or compromise heat stability of whey proteins, depending on pH, ionic strength and type, and co-solute concentration. Protein aggregation may result in opaque appearance and even gelation when proteins form a three-dimension network within a container. These are undesirable qualities to applications such as transparent beverages.
Improving heat stability of why protein for transparent food products such as functional beverages has been a research topic and problem within the food industry for decades. Preheating whey protein, usually at 80-90° C. for 15 min or longer, at neutral pH and a low ionic strength produces filament-like aggregates (also called polymers in some papers). Preheated whey protein has much enhanced heat stability at neutral pH when reheated, resulting from some irreversible physical and chemical bonds. The denatured protein can be used to produce “cold-set gels” that are formed after addition of salt or acidification without additional heating.
It is also known that preheating or denaturation by a reducing agent such as dithiothreitol exposes amino acids originally embedded in globular structures of native α-lactalbumin and β-lactoglobulin, and this allows the cross-linking by transglutaminase (TGase). Excellent heat stability of enzymatically cross-linked whey protein at a concentration of 1% or lower was observed more than two decades ago and was later repeated by others. However, heat stability at a protein content of 5% or higher however remained a challenge, especially when added with salt.
High protein beverages have an average of 6% protein (Vardhanabhuti et al., 2009). Several strategies to improve heat stability of whey protein at relatively high concentrations have been developed. Caseins are chaperones that can reduce the degree of protein unfolding, aggregation, and precipitation. 2% w/v of high purity (>90%) β-casein was observed to be capable of stabilizing 6% w/v β-lactoglobulin solutions at pH 6.0 without salt addition during heating at 90° C. for a period as long as 90 min (Yong and Foegeding, 2009). Lower purity β-casein however was not able to stabilize β-lactoglobulin. Addition of dextran sulfate at an approximate ratio to β-lactoglobulin (6% w/v) improved heat stability of protein at pH 5.6-6.2 when there was no NaCl (Vardhanabhuti et al., 2009). However, all samples formed gels when the ionic concentration and was increased to 30 mM at pH 6.0. Our recent work (Zhang and Zhong, 2009, 2010) utilized microemulsions as templates to form nanoparticles of whey protein by heat or sequential enzymatic cross-linking and heat. The produced nanoparticles had much improved stability when dispersed at 5% w/v in 50 mM sodium phosphate buffer at pH 6.8 and 100 mM NaCl. The production capacity for nanoparticles however was limited.
The subject application provides a solution to the problem of improving heat stability of proteins, such as whey protein, for use in transparent food products such as functional beverages and provides protein-carbohydrate (e.g., whey protein-carbohydrate) conjugates that are heat stable at high protein content (e.g. around 15% (w/v) in both the presence and absence of salt.